This invention pertains to fish vaccines, particularly to certain live-attenuated bacterial vaccines against fish pathogens.
Immune responses to live vaccines are generally of greater magnitude and of longer duration than those produced by killed or subunit vaccines. A single dose of a live-attenuated vaccine can provide better protection against later infection by the wild-type organism, because the attenuated organism persists and metabolizes within the host, and in some cases may replicate in the host for a time. See, e.g., M. Roberts et al., xe2x80x9cSalmonella as Carriers of Heterologous Antigens,xe2x80x9d pp. 27-58 in O""Hagan (ed.), Novel Delivery Systems for Oral Vaccines (1994). Live vaccines better elicit cell-mediated immune responses, which can have a crucial role in controlling infections by intracellular pathogens. Injectable vaccines are impractical in most commercial fish culture due to extensive pond or cage production techniques, large numbers of individual animals, and low value per individual animal. Prior immersion or oral delivery of killed vaccines to fish has yielded inconsistent results. The invasion, persistence, and replication of live-attenuated vaccines has the potential to provide effective, inexpensive vaccines. R. Thune et al., xe2x80x9cStudies on Vaccination of Channel Catfish, Ictalurus punctatus, against Edwardsiella ictalurixe2x80x9d pp. 11-23 in D. Tave et al. (ed.), Recent Developments in Catfish Aquaculture (1994).
An auxotrophic bacterium is a nutritional mutant requiring one or more growth factors to survive and replicate. Certain nutrients have limited availability in vertebrate tissues. A bacterium from an otherwise pathogenic species will be attenuated if it is made auxotrophic for such a limited nutrient. These auxotrophic mutants are potentially useful as live-attenuated vaccines.
Roberts et al. (1994) reviews the use of live-attenuated, transformed Salmonella as potential vectors for vaccinating humans and other mammals orally with heterologous antigens derived from other pathogens. Attenuated strains have been produced by a variety of routes, including strains with aroA or purA mutations. (The aroA gene encodes an enzyme needed in the biosynthesis of aromatic amino acids; and the purA gene encodes an enzyme needed in the biosynthesis of adenine.) See also C. Hornaeche, xe2x80x9cLive Attenuated Salmonella Vaccines and Their Potential as Oral Combined Vaccines Carrying Heterologous Antigens,xe2x80x9d J. Immunol. Meth., vol. 142, pp. 113-120 (1991); D. Sigwart et al., xe2x80x9cEffect of a purA Mutation on a Efficacy of Salmonella Live-Vaccine Vectors,xe2x80x9d Infection and Immunity, vol. 57, pp. 1858-1861 (1989); and S. Hoiseth et al., xe2x80x9cAromatic-Dependent Salmonella Typhimurium are Non-Virulent and Effective as Live Vaccines,xe2x80x9d Nature, vol. 291, pp. 238-239 (1981). In mammalian hosts, however, adenine auxotrophic Salmonella purA mutants are less effective as vaccines than aroA mutants, possibly because purA mutants are overly attenuated due to the extremely low availability of adenine in mammalian tissues.
The channel catfish (Ictalurus punctatus) is the most important aquaculture species in the United States. R. Thune, xe2x80x9cBacterial Diseases of Catfish,xe2x80x9d Chapter 57 (pp. 511-520) in Stoskopf, M. K. (ed.), Fish Medicine (1993) reviews the major bacterial diseases encountered in commercial catfish aquaculture, the most serious of which is enteric septicemia of catfish (ESC). Edwardsiella ictaluri, the bacterium that causes ESC, was first described in 1979 after isolation from catfish farms in Georgia and Alabama. Since then it has been reported in every state that produces channel catfish commercially. Edwardsiella ictaluri was isolated from 46.2% of the channel catfish cases submitted to aquatic animal diagnostic laboratories in Alabama, Louisiana, and Mississippi during 1987-89.
The various Edwardsiella ictaluri strains that have been examined to date have been serologically and biochemically homogenous. As a result, killed bacterins have been evaluated as vaccines against ESC. A protective response has been inconsistent in field trials using killed preparations, and it has been suggested that prior, sub-clinical exposure of vaccinated fish to E. ictaluri during periods in which temperatures were not conducive to disease may have been an important factor in establishing this response; and that a similar response might not be seen in naive fish without a similar sub-clinical exposure. Thune et al. (1994). A variety of preparations were found to stimulate antibody production in these studies, but a positive antibody response did not always correlate to protective immunity unless very high titers of antibody were achieved. Protection of laboratory-reared E. ictaluri-free fish has not been demonstrated and no commercial vaccines for ESC are currently available.
A strong cell-mediated immune response could provide a more effective vaccination against ESCxe2x80x94both for the above reasons, and because E. ictaluri is a facultative intracellular pathogen.
Injection of a killed preparation with an adjuvant is one way to stimulate cell-mediated immunity (CMI), but because of the large numbers, small size, and low economic value of individual fish, this route of vaccination is not practical in commercial catfish production. Live-attenuated strains of pathogenic bacteria could potentially generate a strong CMI. In addition, attenuated strains of invasive pathogens may be delivered via oral and immersion routes, making their administration more economical. However, no previous vaccines have been reported to stimulate cell-mediated immunity against E. ictaluri. 
Commercial farming of hybrid striped bass (Morone saxatilis x Morone chrysops) is a rapidly expanding aquaculture industry in the United States, the Mediterranean region, and southeast Asia, including Taiwan. In the United States, hybrid striped bass production increased from 3750 tons in 1994 to 7000 tons in 1996 (Hybrid Striped Bass Growers Association, personal communication). This fish is adapted for culture in both fresh and brackish water, resulting in the development of significant production of this hybrid species in coastal areas worldwide. In the United States, coastal hybrid striped bass farms are located in Louisiana, Texas, and Florida. In addition, United States producers ship millions of fry and fingerlings annually to marine and brackish water mariculture farms in Taiwan and in the Mediterranean region.
Along with the growth of this industry in coastal areas has come the emergence of the bacterial disease agent Pasteurella piscicida, which has seriously restricted the expansion of commercial aquaculture in warm water coastal areas. (Pasteurella piscicida has recently been renamed Photobacterium damsela subspecies piscicida. The historical nomenclature Pasteurella piscicida is used here.) Pasteurellosis was relatively unknown outside of Japan prior to 1990. In Japan pasteurellosis has caused losses in excess of $20 million annually in cultured yellowtail. The recent growth of coastal aquaculture in the United States and in the Mediterranean region has created ideal conditions for this highly pathogenic, halophilic organism. In Louisiana alone, 32 cases of heavy mortality in coastal hybrid striped bass farms have been reported in the last five years (Louisiana Aquatic Animal Diagnostic Lab case records), with two farms closing as a result of P. piscicida losses.
The gilthead seabream Sparus aurata, and seabass Dicentrarchus labrax, species that are farmed in Israel, Europe, and the Mediterranean, are also highly susceptible to P. piscicida. Production of hybrid striped bass, seabream, and seabass throughout the Mediterranean region is estimated to be tens of thousands of tons annually. P. piscicida has become a serious problem throughout the region.
Pasteurellosis is an acute, rapidly developing disease. Antibiotic treatments have often been impractical or ineffective. In addition, P. piscicida has quickly developed resistance to certain antibiotics. An effective vaccine would circumvent these problems. However, previous vaccinations of hybrid striped bass against P. piscicida using killed autogenous bacterins, Alpharma (Bellevue, Wash.) and AquaHealth (Ontario, Canada), delivered by immersion or injection, have not provided satisfactory results in the field (Dr. R. Ariav, personal communication).
Known host fishes of Pasteurellosis include the following: the temperate basses (Family Percichthyidae), including the white bass Morone americanus, the striped bass Morone saxatilis, and their hybrids; the sea basses (Family Serranidae), including the Japanese sea bass Lateolabrax japonicus, the Asian sea bass Lates calcarifer, and the European sea bass, Dicentrarchus labrax; the jacks (Family Carangidae), including yellowtail Seriola quinqueradiata and striped jack Pseudocaranx dentex; the filefishes (Family Balistidae), including the oval filefish Navodan modestus; and the seabream (Family Sparidae), including the black seabream Acanthopagrus shlegeli, the red sea bream Pagrus major, and the gilthead seabream Sparus aurata. 
R. Kusuda et al., xe2x80x9cThe Efficacy of Attenuated Live Bacterin of Pasteurella piscicida against Pseudotuberculosis in Yellowtail,xe2x80x9d Bull. Eur. Assoc. Fish Pathol., vol. 8, pp. 50-52 (1988) discloses that a degree of protective immunity was conferred by immersion vaccination of yellowtail with a Pasteurella piscicida strain that had been attenuated by serial passages on Brain Heart Infusion agar. These authors examined the response of yellowtail to formalin killed (FKB), heat killed (HKB), and the live-attenuated (ALB) bacterins, and found that the ALB reduced mortality to challenge from 81.3% in controls to 25.3% in vaccinated fish. The FKB and HKB reduced mortality to 57.3% and 78.7%, respectively. In addition, ALB increased phagocytic activity over controls from 4.0% to 19.0%, compared to 4.8% with HKB and 8.0% with FKB, while the increase in antibody level was similar for all three treatments. The authors stated that these results indicated that protection from P. piscicida infection may have been based on activation of phagocytes. The ALB vaccines of this study, however, were produced by serial passage on growth media, and are thus potentially susceptible to spontaneous reversion to virulence. The overall genetic change needed for reversion in such cases can be quite lowxe2x80x94even a single point mutation may suffice.
U.S. Pat. No. 5,536,658 discloses a chondroitinase-attenuated Edwardsiella strain used as vaccine for catfish and other fish susceptible to Edwardsiella infection, administered by immersion, injection, or in feed.
U.S. Pat. No. 5,498,414 discloses attenuated Aeromonas salmonicida strains used as immersion vaccines for chinook salmon and rainbow trout. The attenuated strains were reported to lack a functional A-protein, a component of the cell membrane. The A-protein gene could be disrupted, for example, by insertion of a gene encoding an antigenic protein of another fish pathogen, thus potentially allowing the attenuated Aeromonas salmonicida to vaccinate fish against two pathogens.
E. Dunn et al., xe2x80x9cVaccines in Aquaculture: The Search for an Efficient Delivery System,xe2x80x9d Aquacultural Engineering, vol. 9, pp. 23-32 (1990) reviews various vaccine delivery methods for aquaculture.
P. Homchampa et al., xe2x80x9cConstruction and Vaccine Potential of an aroA mutant of Pasteurella haemolytica,xe2x80x9d Veterinary Microbiology, vol. 42, pp. 35-44 (1994) discloses the use of an attenuated Pasteurella haemolytica mutant with an aroA mutation to immunize mice, as a model for a cattle vaccine against bovine pneumonic pasteurellosis.
L. Vaughan, xe2x80x9cAn Aromatic-Dependent Mutant of the Fish Pathogen Aeromonas salmonicida Is Attenuated in Fish and Is Effective as a Live Vaccine against the Salmonid Disease Furunculosis,xe2x80x9d Infection and Immunity, vol. 61, pp. 2172-2181 (1993) discloses that an attenuated Aeromonas salmonicida with an aroA mutation was not virulent when injected intramuscularly into Atlantic salmon; and that intraperitoneal vaccination with the attenuated strain conferred protective immunity to brown trout against infection by a virulent A. salmonicida strain. See also L. Vaughan et al., xe2x80x9cField Testing of a Novel Live-Attenuated Furunculosis Vaccine in Atlantic Salmon (Salmo salar), in Book of Abstracts, Biotechnological Approaches to the Culture and the Diseases of Fish and Shellfish (Cork, a Ireland, 14-17 September 1992).
C. Lobb, xe2x80x9cSecretory Immunity Induced in Catfish, Ictalurus punctatus, Following Bath Immunization,xe2x80x9d Developmental and Comparative Immunology, vol. 11, pp. 727-738 (1987) discloses that catfish developed a mucosal immune response when immersed in an antigen bath containing dinitrophenylated-horse serum albumin, but that few of the catfish developed a humoral response.
J. Plumb et al., xe2x80x9cVaccination of Channel Catfish, Ictalurus punctatus (Rafinesque), by Immersion and Oral Booster against Edwardsiella ictaluri,xe2x80x9d J. Fish Diseases, vol. 16, pp. 65-71 (1993) discloses a formalin-killed Edwardsiella ictaluri immersion vaccine that produced humoral immunity in Ictalurus punctatus, with or without a subsequent oral booster.
R. Thune et al., xe2x80x9cStudies on Vaccination of Channel Catfish, Ictalurus punctatus, against Edwardsiella ictalurixe2x80x9d pp. 11-23 in D. Tave et al. (ed.), Recent Developments in Catfish Aquaculture (1994) discloses a formalin-killed Edwardsiella ictaluri immersion vaccine for the catfish Ictalurus punctatus, with or without a subsequent oral booster.
We have discovered effective live-attenuated vaccines against Edwardsiella ictaluri. We have also discovered effective live-attenuated vaccines against Pasteurella piscicida. Both vaccines are incapable of reversion to virulence. Both were made by large deletion mutations either in the aroA gene or in the purA gene.
We have also discovered that these vaccines may be used not only to vaccinate fish against Edwardsiella ictaluri or Pasteurella piscicida, but also to serve as vectors to present antigens from other pathogens to the fish immune system, thereby serving as vaccines against other pathogens as well, with no risk of infection by reversion to the virulent form of the pathogen in which the antigen occurs naturally.